Infection of humans with HIV-1 and monkeys with SIV is frequently accompanied by a variety of neurological disease manifestations. Studies from HIV-1 and SIV strongly argue that genetic changes in viruses occur during the course of infection and give rise to mutant viruses that are advantaged for replicating in specific cell types. These observations suggest that mechanisms by which HIV-1 and SIV induce neurological diseases can potentially be understood by studying the biologic and genetic characteristics of viruses that adapt to replication in brain. We have shown that the SIVmac155/NT variant isolated from encephalitic brain of a rhesus macaque has changes in the genetic code of its external envelope glycoprotein gene (gp120) that determine replicative potential in brain microglial cells. This is consistent with previous studies defining gp120 as the genetic determinant of cell tropism for HIV-1 and SIV. To evaluate the importance of other viral domains for virus replication in microglial cells, we constructed a cloned SIV recombinant, called N/3' recombinant, that is fully-competent to replicate in microglial cells by exchanging viral sequences spanning from gp120 to the 3'LTR of SIVmac239 with those derived from the NT variant. The viral domains that contribute to virus replication in M and microglial cells were more precisely delineated by construction of finer recombinant viruses and site-specific mutants. Our data indicate that the gp41 and nef gene are required for efficient virus replication in M ; however, only the nef gene can enhance virus replication in microglial cells. Interestingly, the nef gene did not enhance virus replication in rhesus macaque lymphocytes and human T-lymphocyte cell lines. These data indicate that the positive effect of the nef gene on virus replication is specific to macrophage lineage cells, especially microglial cells. Mutations in the nef gene not overlapping with V3 of the 3'LTR were responsible for this activity, and they were specifically present in the NT variant, but not in other SIVs with no or low replicative capacity in microglial cells. Therefore, the mutation in the nef gene enhancing virus replication in microglial cells evolved specifically in the microglial cell-tropic NT variant.